高致病性猪生殖与呼吸综合征病毒感染猪外周血单核细胞cDNA文库的构建

Construction of cDNA library of peripheral blood mononuclear cells of pigs inoculated with highly pathogenic porcine reproductive and respiratory syndrome virus

为应用CloneminerTMcDNA Library Construction Kit构建高致病性猪生殖与呼吸综合征病毒(PRRSV)感染猪的外周血单核细胞cDNA文库,以1×105TCID50PRRSV肌肉接种健康猪,攻毒后第3、5、7、9、14、21及28 d分别采集感染猪外周血,分离单核细胞,提取总RNA、mRNA,以mRNA为模板,通过引物Biotin-attB2-Oligo(dT)、SuperScript.ⅡRT合成5′末端具有attB1接头的cDNA。用柱层析方法纯化cDNA,纯化后的cDNA与pDONRTM222载体进行BP重组,于大肠杆菌DH10B转化,进行文库容量测定。结果表明,构建的高致病性PRRSV感染猪外周血单核细胞cDNA文库的库容量为1.36×107CFU,平均插入片段长度大于1 200 bp,重组率为94.3%,为进一步从文库中筛选未知细胞因子及相关基因提供了有效的工具。

To construct a cDNA library of peripheral blood mononuclear cells of pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus（PRRSV） by CloneminerTM cDNA Library Construction Kit,healthy pigs were inoculated with 105TICD 50 PRRSV,then the peripheral blood from experimentally infected pigs were collected on day 3,5,7,9,14,21 and 28 post-inoculation.The mononuclear cells were isolated from the peripheral blood.The total RNA and mRNA were extracted.The ligated Adapter to the 5′ end of cDNAs were synthesized by Biotin-attB2-Oligo（dT） primers and SuperScript.Ⅱ RT.The cDNAs were isolated by column chromatography method and inserted into pDONRTM222 vector,and they were transformed into Escherichia coli DH10B.The results indicated that the titre of the constructed cDNA library was 1.36×107 CFU,the average length of the inserts was more than 1 200 bp and the recombination rate was 94.3%.The result was helpful for screening unknown cytokine and relative genes from the library.